Abstract

Recent developments in antibody-based immunotherapy, especially targeting immune checkpoints, have revolutionized cancer treatment. Immune checkpoints are inhibitory pathways responsible for dampening the immune response in order to ensure self-tolerance under physiological conditions. In cancer cells, however, immune checkpoints are often utilized as a mechanism to escape the immune system. Numerous studies have demonstrated that blocking immune checkpoints restores potent anti-tumor immune responses and ultimately leads to the elimination of cancer cells. Accordingly, several monoclonal antibodies (mAbs) targeting immune checkpoint receptors are currently in the market. The CD47-SIRPα myeloid-specific immune checkpoint controls the immune response by negatively regulating phagocytosis. SIRPα, expressed on phagocytic cells, triggers a negative signal upon binding to CD47, ubiquitously expressed on healthy cells and overexpressed on several cancer types. Hence, the blockade of the CD47-SIRPα signaling pathway constitutes a promising approach to mediate phagocytosis of tumor cells. This blockade, however, may also induce unwanted toxicity to healthy cells, which also express CD47. In order to reduce systemic toxicity while promoting the elimination of tumor cells, the blockade of the CD47-SIRPα immune checkpoint should be restricted to cancer cells. Acute myeloid leukemia (AML) is a severe hematological cancer with a five year survival rate of 25%. Furthermore, while immunotherapies are already in clinical use for other hematological diseases, chemotherapy remains the first-line treatment for AML. This indicates an urgent need for the development of new and effective approaches that offer a better prognosis to AML patients. In order to provide a novel therapeutic strategy, we generated local inhibitory checkpoint molecules, which are antibody derivatives that deliver the benefits of blocking the CD47-SIRPα immune checkpoint to AML cells. The disruption of the myeloid-specific immune checkpoint is achieved by the endogenous SIRPα domain, which is genetically fused to an antibody fragment targeting the AML antigen CD33. Since the physiologically low affinity of SIRPα to CD47 prevents it from targeting CD47 by itself, the binding of the local inhibitory checkpoint molecules is dictated by the high affinity CD33-binding domain. Consequently, the anti-tumor effects of these molecules are confined to CD33-expressing AML cells. In order to investigate the best strategy to block the CD47-SIRPα immune checkpoint on AML cells, three different local inhibitory checkpoint molecules were created: local inhibitory checkpoint mAbs (licMABs), single-arm licMABs (licMABssingle) and local inhibitory checkpoint antibody derivatives (liCADs). All three formats bind CD33 with high affinity, disrupt the CD47-SIRPα axis by the endogenous SIRPα domain and activate immune effector cells. They diverge, however, in the binding valency to CD33 and the immune effector cell-activating domain. LicMABs target CD33 with both antibody arms, while licMABssingle and liCADs target CD33 monovalently. Moreover, licMABs and licMABssingle activate effector cells by an IgG1 Fc domain and liCADs contain a single chain variable fragment (scFv) activating uniquely CD16. The in vitro evaluation of these molecules confirmed the preferential binding to CD33-expressing cells even in the presence of a large antigen sink created by CD47 expressed on healthy cells. In addition, all local inhibitory checkpoint molecules induced Natural Killer (NK) cell-mediated lysis of AML cells and licMABs and licMABssingle, but not liCADs, enhanced phagocytosis of AML cell lines and primary, patient-derived AML cells. Importantly, we determined that the expression levels of CD47 on primary AML cells influence the outcome of licMAB-mediated phagocytosis. In summary, this work establishes licMABs as a promising strategy to block the CD47-SIRPα immune checkpoint on high CD47-expressing AML cells. Furthermore, the marginal binding of these molecules to CD47 on healthy cells ensures a highly specific immune response and lowers the risk of unwanted side effects.